1. Field of the Invention
The present invention relates to the electrochemistry of enzymes, in particular the electrochemistry of hydroxylase or monooxygenase enzymes.
2. Description of the Background
Hydroxylases are known as catalysts for the oxidation of various substrates. For example, methane monooxygenase is an efficient catalyst for the oxidation of methane by molecular oxygen to give methanol as the sole product.
Studies of the structure and mechanisms of electrochemistry of hydroxylases have been reported in the literature.
There are two forms of the enzyme methane monooxygenase, soluble (sMMO) and particulate (pMMO). The soluble enzyme from Methylococcus capsulatus (Bath) consists of an hydroxylase (Mw 250.5 kDa), a reductase (Mw 38.5 kDa) and a regulatory component, protein B (Mw 15.9 kDa) all of which are required for activity. The hydroxylase is made up of two protomers in an a2b2g2 arrangement and the X-ray crystal structure of this component has been solved. The sMMO from Methylosinus trichosporium OB3b has a very similar composition and has also been well characterised. The active site in sMMO is a di-iron centre, bridged by an hydroxo group (in the resting enzyme), which resides in the a subunit of the hydroxylase. Reducing equivalents from NADH are transferred to active site through F.sub.2 S.sub.2 and FAD centres in the reductase. Protein B contains no metal ions or cofactors and the details of its regulatory role are unclear. In the resting enzyme the irons are in the fully oxidised Fe(III)Fe(III) state. There are two other oxidation states readily available to the di-iron cluster, namely, the mixed valent Fe(III)Fe(II) and the fully reduced Fe(II)Fe(II) states. It is the Fe(II)Fe(II) form of the hydroxylase which reacts with and activates O.sub.2 during enzyme turnover.
The redox potentials of the di-iron centres in M. capsulatus (Bath) and M. trichosporium OB3b have been measured in three independent studies. The (electrode potential) values for the Fe(III)Fe(III)/Fe(III)Fe(II) and Fe(III)Fe(II)/Fe(II)Fe(II) couples have been the subject of some debate. The redox properties of the hydroxylase component of soluble methane monooxygenase from the two different organisms have been extensively investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Indirect titration of the hydroxylase di-iron centres with redox active mediators were employed in these studies. The concentrations of the mixed valent and fully reduced hydroxylase species were determined by EPR (electron-proton resonance) or EPR and Moessbauer spectroscopy at very low temperatures (4.2-18.degree. K).
In the field of protein electrochemistry, thiol- or disulfide-containing organic molecules have been found to be particularly good modifiers because they chemisorb through a strong gold-surfer bond thereby giving a stable layer of surface coverage on the electrode. Relevant amino acids may also be chemisorbed in this fashion and thus promote protein electrochemistry at the electrode surface. The use of cysteine containing peptides which also contain functional amino acids (e.g. arginine, lysine, histidine), as promoters for protein electrochemistry has been investigated.
However, contrary to the field of protein electrochemistry, it is not a trivial task to carry out direct electrochemical measurements without the aid of mediators, on redox enzymes. A major difficulty is that often the redox centres are buried deep within the protein, far from the surface, so that the distance electrons must traverse to an electrode can be large enough to reduce the rate of electron transfer to a negligibly small value. Also as most redox enzymes are much larger and structurally less rigid than non-redox proteins, they are more liable to deformation and lose of activity on electrode surfaces.
The electrochemistry of hydroxylases in the presence of a mediator is complicated by the possibility of interaction between the hydroxylase and the mediator, while the mediator may disturb the determination of species concentrations at temperatures different from those at which the redox reactions are carried out.